Background: Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) via Ser108 of the AMPK β1 subunit, a mechanism that is distinct from metformin, which increases AMP:ATP. Many cancers have high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is unknown if targeting the AMPK-ACC-lipogenic pathway using salicylate and metformin may be effective for inhibiting cancer cell survival. Results: Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations of aspirin. These clinically achievable concentrations of salicylate activated AMPK per the increasing phosphorylation of ACC and suppressing the activity of mTOR effectors kinase p70-S6 kinase and S6; effects that were enhanced with the addition of metformin and blunted in mouse embryonic fibroblasts (MEFS) deficient in AMPK β1. MEF cells deficient in AMPK β1 were more resistant to salicylates inhibitory effect on proliferation. Supplementation of media with fatty acids and mevalonate reverses the suppressive effects on cell survival indicating the inhibition of de novo lipogenesis is likely important. Conclusions: Salicylate increases ACC phosphorylation, reduces phosphorylation of mTOR targets and inhibits de novo lipogenesis in prostate and lung cancer cells, with concentrations of salicylate achievable through the ingestion of Aspirin (0.25-1.0mM) these effects are blunted in AMPK β1 deficient cells. Effects on AMPK activity via ACC phosphorylation as well as reductions in mTOR signalling targets and de novo lipogenesis are enhanced when used in combination with metformin. Suppressive effects on prostate and lung cancer cell survival are ameliorated when media is supplemented with mevalonate and fatty acids. Pre-clinical studies evaluating the use of salicylates alone and with metformin to inhibit de novo lipogenesis and the growth of prostate and lung cancers are warranted. / Thesis / Master of Science (MSc)

Implication du PTP dans la mort cellulaire β pancréatique L'hyperglycémie, l'hyperfructosémie et l'ischémie-reperfusion sont délétères pour la viabilité cellulaire β pancréatique, jouant un rôle majeur dans la perte de la masse cellulaire β. Le pore de transition de perméabilité mitochondriale (PTP) est un canal mitochondrial impliqué dans le déclenchement de la mort cellulaire. Des données récentes montrent l'implication du PTP et du stress oxydant dans la toxicité induite par l'ischémie-reperfusion sur cardiomyocytes et également dans la glucotoxicité induite sur cellules endothéliales. La première partie de notre étude a visé à étudier l'implication de l'ouverture du PTP dans la mort cellulaire des cellules INS-1 et des îlots pancréatiques humains soumis à de fortes concentrations de glucose et de fructose. Nous démontrons que l'incubation des cellules INS-1 et des îlots pancréatiques humains en présence de 30 mM de glucose ou 2,5 mM de fructose déclenche une ouverture du PTP et induit la mort cellulaire. La metformine et la Cyclosporine A (CsA) préviennent l'ouverture du PTP et la mort cellulaire induite par le glucose et le fructose. La deuxième partie de notre travail montre que l'exposition des INS-1 à une heure de carence en substrat concomitante d'une hypoxie, suivie d'une restauration des conditions basales conduit à l'ouverture du PTP et à une majoration drastique de la mort cellulaire. Ces deux évènements sont totalement prévenus par l'incubation préalable par la CsA et la metformine mais aussi par la N-Acétyl-Cystéine (NAC) ou par l'exposition à une anoxie, soulignant ainsi le rôle fondamental du stress oxydant dans le déclenchement de l'ouverture du PTP et de la mort cellulaire. Nous montrons qu'au cours de l'ischémie-reperfusion simulée, la production de superoxide est bi-phasique : nous décrivons un premier pic de production au cours de la carence en substrat, lié à un flux reverse d'électrons au sein du complexe I de la chaîne respiratoire. Ce premier pic est suivi d'un deuxième pic de production après la restauration du niveau de substrats et d'O2, lié à l'ouverture du PTP. La NAC, l'anoxie ou la metformine préviennent les deux pics de production de superoxide tandis que la CsA prévient seulement le second pic. Enfin, nous montrons que l'hypoxie seule n'induit ni stress oxydant, ni ouverture du PTP ni mortalité cellulaire. L'ensemble de notre travail démontre le rôle central du PTP dans la gluco-fructotoxicité et dans la toxicité induite par l'ischémie-reperfusion sur la cellule β pancréatique. Ainsi, prévenir l'ouverture du PTP peut-être une approche intéressante pour préserver la viabilité cellulaire β. / PTP involvement in β pancreatic cell death Hyperglycemia, hyperfructosemia and ischemia-reperfusion play a major role in the progression of β cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. PTP opening and oxidative stress have been shown to be involved in ischemia-reperfusion injury on cardiomyocytes and in hyperglycemia-induced cell death in endothelial cells. In the first part of this work, we have examined the involvement of PTP opening in INS-1 cells and human pancreatic islets cell death induced by high levels of glucose or fructose. We first reported that Metformin and Cyclosporin A (CsA) prevented Ca2+-induced PTP opening in permeabilized and intact INS-1 cells. We then shown that incubation of INS-1 cells and human islets in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. Because both Metformin and CsA prevented glucose and fructose induced PTP opening, and hampered glucose and fructose induced cell death, we conclude that PTP opening is involved in high glucose and high fructose induced INS-1 and human islets cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β cell viability. In the second part of the work, we demonstrate that the incubation of INS-1 cells in the absence of energy substrates in hypoxic condition for 1 hour followed by incubation in normal condition led to PTP opening and to a dramatic increase in cell death. Both events were totally prevented when PTP opening was inhibited by either Cyclosporin A (CsA) or Metformin or when the cells were incubated in the presence of the antioxidant N-acetyl-cystein (NAC), in anoxia, highlighting the implication of oxidative stress is the commitment of PTP opening. Superoxide production increased during the removal of energy substrates, due to reverse electron flux through complex I and again increased when normal energy substrate and O2 were restored, due to PTP opening. NAC, anoxia or Metformin prevented the two phases of oxidative stress, while CsA prevented only the second one. Hypoxia alone did not induce oxidative stress, PTP opening or cell death. Our work demonstrates the implication of PTP opening in ischemia-reperfusion injury and gluco- fructotoxicty in β pancreatic cells. We therefore suggest that preventing PTP opening might be a new approach to preserve β cell viability.

Parkinsons sjukdom (PD) är ett tillstånd som ger en försvårad och försämrad livskvalité. I dagsläget finns det endast symtomatiska läkemedel men ingen bot med vilken sjukdomen upphör eller som bromsar förloppet. Pågående forskningsarbete utgår bland annat från att ta fram nya läkemedel men även också undersöka om redan befintliga läkemedel går att använda som behandling av PD. Många av de redan befintliga läkemedlen som testas är de som har förmågan att påverka proteinet α-synuklein (α-syn) och dess aggregering, som visats vara en central orsak till uppkomsten av PD. I föreliggande litteraturstudie undersöktes på vilket sätt en del läkemedel vars indikation är PD och även andra sjukdomstillstånd såsom astma, påverkar (ex. påskyndar eller inhiberar) in-vitro aggregering av α-syn. Därutöver genomfördes en detaljerad analys av de utvalda läkemedlen och deras effekt på α-syn aggregering utifrån deras kemiska egenskaper med avseende på löslighet (hydrofila, lipofila, amfifila) och inbindning till α-syn. Här kunde det visas att aggregering av α-syn inhiberades av alla utvalda läkemedel förutom dexametason, som istället påskyndade aggregeringskinetiken för proteinet. Dessutom uppvisade fasudil, ceftriaxon, dopamin, entakapon och tolkapon inbindning till delar av (hydrofila, hydrofoba eller amfifila) vilka delade samma fysikalkemiska egenskaper som α-syn. Därtill uppvisade utvalda läkemedel med till viss del plana strukturer (ex. aromatiska ringar) direkt inbindning till α-syn, vilka också rapporterats ha en något högre grad av transport över blod-hjärnbarriären, dock måste dessa fynd mer noggrant undersökas. Sammanfattningsvis visade alla utvalda läkemedel förutom dexametason anti-aggregeringsegenskaper (hämmande) mot α-syn genom att antingen indirekt eller direkt binda till proteinet och därmed hindra proteinet från att börja binda till sig själv. Mer studier måste genomföras för att studera effekten av läkemedelsexponering på α-syn för att identifiera viktiga segment av proteinet som kan utgöra läkemedelsmål för inhibering av α-syn aggregering. / Parkinson's disease (PD) is a condition that leads to an aggravated and worsened quality of life. At present, there are only symptomatic drugs for PD but no cure that eradicate the disease nor halter the disease progression have been found. Current research is being carried out to develop new drugs, but efforts also investigate whether existing drugs can be used as treatment for PD. Many of the already existing drugs being tested are those that have the ability to interact with a protein called α-synuclein (α-syn), that has been implicated to be a major player for onset of PD. In the present literature study, it was investigated in what way some drugs, whose indication is PD but also other diseases such as asthma, affect (i.e. propagate or inhibit) the in-vitro aggregation kinetics of αsyn. Additionally, a detailed analysis of the investigated drugs and their effect on the aggregation pathway was made to characterize common chemical features of the selected drugs based upon choice of solvents and binding to α-syn. Here, it could be shown that aggregation of α-syn is inhibited upon exposure to all selected drugs except dexametason which instead propagated aggregation of α-syn. In addition, fasudil, ceftriaxone, dopamine, entacapone and tolcapone was found to bind to parts (hydrophilic, hydrophobic or amphiphilic) of α-syn similar to their solubility features. Moreover, the selected drugs that were found to bind to α-syn seemed to exhibit planar in structure (i.e. aromatic rings) and also be associated to pass the blood-brain barrier to a greater extent, however these findings need to be more thoroughly investigated. In summary, all drugs but dexametason were shown to inhibit aggregation of α-syn invitro by either indirectly or directly affecting the aggregation of the protein. Further investigations need to be carried out to study the effect of drug exposure on α-syn aggregation in order to propose key segments of α-syn that can act as drug targets for inhibition of protein aggregation.

Introdução: A síndrome dos ovários policísticos (SOP) é um distúrbio endócrino complexo com aspectos reprodutivos e metabólicos. Alterações do tecido adiposo participam da fisiopatologia da síndrome e anormalidades do metabolismo de ácidos graxos, bem como o estresse oxidativo, parecem ter papel importante nesse processo. Assim, tanto o exercício físico como a metformina são comumente recomendadas. O objetivo deste estudo foi avaliar a expressão de genes envolvidos com a síntese e oxidação dos ácidos graxos e com os níveis de estresse oxidativo no tecido adiposo de ratas com modelo experimental de SOP tratadas com metformina (Met), submetidas ou não a exercício físico em esteira (Ex). Métodos: Foram utilizadas 46 ratas, Wistar, que receberam no 2º dia de vida, uma única injeção subcutânea de propionato de testosterona (1,25 mg) para a indução de estado de estro permanente e óleo de sésamo (controles). Com 80 dias de idade iniciou-se o experimento e os animais foram divididos em 5 grupos: 1) Controle; 2) SOP ; 3) SOP+Met; 4) SOP+Ex; 5) SOP+Met+Ex.Os tratamentos tiveram duração de 6 semanas. Os animais foram sacrificados aos 120 dias de vida e foram retirados os depósitos das gorduras inguinal e mesentérica. Os depósitos de gordura inguinal e mesentérica foram utilizados para análise da expressão dos genes Acaca, Srebp1, Cpt1 e Cd36, por PCR quantitativo em tempo real, e dos níveis de estresse oxidativo pela dosagem das glutationas reduzida (GSH) e oxidada (GSSG). Adicionalmente, o tamanho dos adipócitos foi analisado por histomorfometria. Resultados: Na análise histomorfométrica dos adipócitos da gordura mesentérica, houve diminuição significativa no grupo SOP+Met+Ex em relação ao grupo SOP. O grupo SOP apresentou hipertrofia em relação ao grupo controle. A análise na gordura inguinal não apresentou nenhuma diferença entre os grupos.Não houve diferença estatística entre os grupos nas análises de expressão gênica das gorduras mesentérica e marrom. Não identificamos diferenças nas medidas de estresse oxidativo (razões GSH/GSSG ,GSH, GSH total, GSSG e razão GSH/GSSG) no tecido adiposo subcutâneo, mesentérico, perigonadal e marrom entre os depósitos de gordura dos animais em nenhum dos grupos avaliados / Introduction: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder, affecting both reproductive and endocrine systems. Changes in adipose tissue are involved in the pathophysiology of the syndrome, and abnormalities of fatty acid metabolism, as well as oxidative stress, appear to play an important role in this process. Thus, both physical exercise and metformin are commonly recommended. The objective of this study was to evaluate the expression of genes involved in the synthesis and oxidation of fatty acids and oxidative stress levels in adipose tissue of in an experimental model of PCOS using Wistar rats. Methods: 46 rats, Wistar, treated with a single subcutaneous injection of testosterone propionate (1.25 mg) for the induction of permanent estrus status (PCOS model) or sesame oil (controls) on the 2nd day of life. Experimental phase of the study was started when animals completed 80 days of age. They were divided into 5 groups: 1) Control; 2) PCOS; 3) PCOS+ Met; 4) PCOS + Ex; 5) PCOS + Met + Ex. Intervention lasted 6 weeks. Animals were sacrificed at 120 days of age and the deposits of inguinal and mesenteric fat were removed. Inguinal and mesenteric fat deposits were used to analyze the expression of Acaca, Srebp1, Cpt1 and Cd36 genes by quantitative realtime PCR and oxidative stress levels by reduced (GSH) and oxidized glutathione (GSSG). In addition, adipocyte size was analyzed by histomorphometry. Results: In the histomorphometric analysis of mesenteric fat adipocytes, there was a significant decrease in the PCOS + Met + Ex group in relation to the PCOS group. PCOS group presented hypertrophy in relation to the control group. Inguinal fat analysis did not present any differences between the groups. There was no statistical difference between groups in analyzes of gene expression of mesenteric and brown fats. We did not identify differences in oxidative stress measurements (GSH / GSSG, GSH, total GSH, GSSG and GSH / GSSG ratio) in the subcutaneous, mesenteric, perigonadal and brown adipose tissue among the fat deposits of the animals in any of the evaluated groups

Objetivamos investigar o papel da quinase ativada por adenosina monofosfato (AMPK) na doença renal crônica. Induzimos nefrite túbulo-intersticial (NTI) em camundongos C57BL/6 e LyzM-cre AMPKflox/flox através de ração com adenina, e tratamos com metformina (Met) 200 mg/kg/dia. Avaliamos ainda o efeito da Met sobre a transição epitélio-mesenquimal (TEM) em células tubulares epiteliais renais murinas (linhagem MM55.K). Os C57BL/6 tratados apresentaram preservação da função renal; maior frequência de macrófagos (MØ) M1, em detrimento dos M2; e redução de marcadores de fibrose. Os LyzM-cre AMPK-/- não diferiram dos LyzM-cre AMPK+/+ quanto à intensidade da lesão, por a molécula já se encontrar infrarregulada na NTI. Contudo, ao serem tratados com Met, os LyzM-cre AMPK+/+ evoluíram melhor do que os não tratados, o mesmo não se verificando nos LyzM-cre AMPK-/-, sugerindo que a ação da Met nos MØ é dependente de AMPK. As MM55.K, após estímulo com Met, exibiram maior captação de glicose, expressão do transportador Glut-2, ativação da glicólise, e resistência à TEM. / We aimed to investigate the role of adenosine monophosphate activated kinase (AMPK) on chronic kidney disease. We induced tubulointerstitial nephritis (TIN) in C57BL/6 and LyzM-cre AMPKflox/flox mice by feeding them adenine diet, and then treating with metformin (Met) 200 mg/kg/day. We also evaluated the effect of Met on epithelium-to-mesenchyma transition (EMT) of murine epithelial renal tubular cells (lineage MM55.K). Met-treated C57BL/6 mice presented preserved kidney function, greater frequency of M1 macrophages (MØ) compared to M2 ones, and reduced markers of fibrosis. Disease severity on LyzM-cre AMPK-/- and AMPK+/+ mice did not differ, since the molecule was already downregulated on TIN. However, by treating them with Met, LyzM-cre AMPK+/+ improved in comparison to the non-treated mice. The same did not happen with LyzM-cre AMPK-/- mice, suggesting that Met effect on MØ is AMPK-dependent. MM55.K cells, after stimulus with Met, showed increased glucose uptake, greater expression of the transporter Glut-2, activation of glycolysis, and resistance to EMT.

INTRODUÇÃO: A metformina tem sido amplamente utilizada no tratamento da síndrome dos ovários policísticos (SOP), porém poucos estudos comparam a metformina e a dieta associada a exercícios físicos. O objetivo deste estudo é comparar parâmetros clínicos e laboratoriais de mulheres com SOP em uso de metformina ou através de dieta e exercícios físicos. MÉTODOS: Foram avaliadas 30 mulheres com SOP, com idades entre 18 a 34 anos, as quais foram divididas em dois grupos: grupo A- tratamento com metformina 1.800mg/dia e grupo B- dieta hipocalórica associada a exercícios físicos. Avaliações clínica e laboratorial foram feitas antes dos tratamentos e a cada três meses, por período de seis meses. RESULTADOS: Não houve diferença significativa entre os tratamentos quanto à regularização do ciclo menstrual (p=0,711), acne (p=0,271), hirsutismo (p=0,146) e índice de massa corpórea (IMC) p=0,328; assim como nas dosagens laboratoriais de LH (p=0,147), FSH (p=0,891), testosterona total (p=0,226) e livre(p=0,455), androstenediona (p=0,066), 17alfa-hidroxiprogesterona (p=0,914), SHBG (p=0,791), colesterol total (p=0,692) e frações, triglicérides (0,291) e nos índices de avaliação de resistência insulínica HOMA-R (p=0,111) e relação glicemia/insulina (p=0,976). Os dois tratamentos apresentaram melhora do ciclo menstrual (76%) e do IMC (p<0,001). A diminuição da circunferência abdominal foi maior no grupo B (p=0,006). CONCLUSÕES: Comparando os dois tratamentos não houve diferença nos parâmetros clínicos e laboratoriais avaliados. Ambos foram eficazes na redução do peso corpóreo e na regularização do ciclo menstrual. / Metformin has been widly used in treatment of polycystic ovary syndrome (PCOS) but only few studies compare metformin with diet and exercises. The aim of this study is to compare clinical and laboratorial parameters of women with PCOS using metformin or under diet and exercises. Methods: Thirty women with PCOS were evaluated with ages between 18- 34 years old, who were divided in two groups: group A- treatment with metformin 1.800mg/day and group B- hypocaloric diet and exercises. Clinical and laboratorial evaluations were done before treatment and each three months during a period of six months. RESULTS: There were no significant differences between treatments for menstrual disturbances (p=0,711), acne (p=0,271), hirsutism (p=0,146) and body mass index (BMI), p=0,328; in laboratorial parameters there were no significant differences for LH (p=0,147); FSH (p=0,891), total testosterone (p=0,226), free testosterone (p=0,455), androstenedione (p=0,066), 17alfa-hidroxiprogesterone (p=0,914), SHBG (p=0,791), total cholesterol (p=0,692) and fractions and indexes for evaluation of insulin resitence HOMA-IR (p=0,111) and glycemia and insulin ratio (p=0,976). Both treatments improved menstrual disturbances (76%) and BMI (p<0,001). The abdominal circunference decreasing was greater in group B (p<0,006). CONCLUSIONS: Comparing both treatments there were no differences in clinical and laboratorial parameters evaluated. Both were efficient in improving body mass index and menstrual disturbances.

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2018-02-15T14:23:39Z No. of bitstreams: 1 marinagobbemoschetta_tese.pdf: 3429562 bytes, checksum: 62a703a94d8ded4bd80d7a77a4476e3d (MD5) / Made available in DSpace on 2018-02-15T14:23:39Z (GMT). No. of bitstreams: 1 marinagobbemoschetta_tese.pdf: 3429562 bytes, checksum: 62a703a94d8ded4bd80d7a77a4476e3d (MD5) Previous issue date: 2016-11-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Introduction: Hypoxia is a known adverse factor to cancer treatment, able to activate the signaling pathways PI3K and AMPK/Akt/mTOR, the two major pathways involved in the angiogenesis process. This process is regulated by many factors, being the most important factors the Hypoxia-Inducible Factor-1α (HIF-1α) and Vascular Endothelial Growth Factor (VEGF). Metformin has demonstrated its ability to reduce the incidence of cancer in diabetic patients with positive results especially in breast cancer, inhibiting cell growth by AMPK/Akt/mTOR signaling pathway. Likewise, LY294002 is the major inhibitor of PI3K/AKT/mTOR signaling pathway that has anti-angiogenic properties, capable to reduce the release of growth factors such as VEGF. The treatment with metformin in addition to the inhibitor LY294002 in both normal oxygen and hypoxia conditions, as well as the action in the process of angiogenesis in canine mammary tumors is unprecedented. Objectives: To evaluate the influence of metformin and LY294002 inhibitor treatment in tumor angiogenesis as a therapeutic strategy in mammary tumors in a in vitro and in vivo study. Material and Methods: In the in vitro study, we analyzed the cell viability of canine mammary tumor cell lines CMT-U229 (benign mixed tumor) and CF41 (metastatic carcinoma) before and after treatment with metformin and/or LY294002 by MTT assay. The cell cycle distribution was analyzed by flow cytometry flow, protein and gene expression of VEGF and HIF-1α by immunohistochemistry and RT-PCR, respectively. All experiments were performed in normal oxygen conditions and under hypoxia by the addition of cobalt chloride (CoCl2). For the in vivo study, CF41 cells were implanted in 20 female mice BALB / c nude nude. After 28 days of treatment with metformin and LY294002, it was analyzed micro vessel density by CD31 expression by immunohistochemistry as well as gene and protein expression of HIF-1α and VEGF in the tumor tissue. Results: The treatment with metformin and LY294002 was able to reduce the cellular viability after 24 hours. The protein and gene expression of HIF-1α and VEGF decreased after treatment with metformin and LY294002, both in normal oxygen conditions and hypoxia. In the in vivo study, there was a decrease in tumor size, protein and gene expression of HIF-1α and VEGFA, in addition to the decreasing of CD31 expression after all treatments. Conclusions: Our results demonstrated the effective action of metformin and LY294002 inhibitor in controlling the angiogenesis process in mammary tumors by the angiogenic factors VEGF and HIF-1α. / Introdução: A hipóxia é um conhecido fator adverso ao tratamento do câncer, capaz de ativar as vias de sinalização PI3K e MAPK/Akt/mTOR, as duas principais vias envolvidas no processo de angiogênese. Esse processo é regulado por inúmeros fatores, sendo os mais importantes o Fator Induzido por Hipóxia-1α (HIF-1α) e o Fator de Crescimento Endotelial Vascular (VEGF). A metformina tem demonstrado sua capacidade de diminuir a incidência de neoplasias em pacientes diabéticos com resultados positivos especialmente no câncer de mama, inibindo o crescimento celular através da via de sinalização MAPK/Akt/mTOR. Da mesma forma, o LY294002 é um importante inibidor da via de sinalização PI3K/AKT/mTOR que possui propriedades anti-angiogênicas, capaz de diminuir a liberação de fatores de crescimento como o VEGF. A combinação do tratamento com metformina e com o inibidor LY294002 tanto em condições normais de oxigênio quanto em hipóxia, bem como a ação no processo de angiogênese em tumores mamários caninos é inédita. Objetivos: Avaliar a influência do tratamento com metformina e com o inibidor LY294002 na angiogênese tumoral como estratégia terapêutica nas neoplasias mamárias, em um estudo in vitro e in vivo. Material e Métodos: No estudo in vitro, foi analisada a viabilidade das células das linhagens tumorais mamárias caninas CMT-U229 (tumor misto benigno) e CF41 (carcinoma metastático) antes e após o tratamento com metformina e/ou LY294002 por ensaio MTT. A distribuição das células nas fases do ciclo celular foi avaliada por citometria de fluxo, a expressão proteica e gênica do VEGF e do HIF-1α por imuno-histoquímica e PCR em Tempo Real, respectivamente. Todos os experimentos foram realizados em condições normais de oxigênio e sob hipóxia pela adição de cloreto de cobalto (CoCl2). Para o estudo in vivo, células da linhagem tumoral mamária canina CF41 foram implantadas em 20 camundongos fêmeas Balb/c nude atímicos. Após 28 dias de tratamento com metformina e LY294002, foi analisada a microdensidade vascular pela expressão do CD31 por imunohistoquímica, bem como a expressão proteica e gênica do VEGF e HIF-1α no tecido tumoral. Resultados: O tratamento com metformina e com LY294002 foi capaz de diminuir significativamente a viabilidade celular, após 24 horas. A expressão proteica e gênica do HIF-1α e do VEGF diminuiu após o tratamento com metformina e LY294002 tanto em hipóxia quanto em condições normais de oxigênio. No estudo in vivo, houve diminuição do tamanho tumoral e da expressão proteica e gênica do VEGFA e HIF-1α, além da diminuição da expressão do CD31, após os tratamentos. Conclusões: Nossos resultados demonstram a efetiva ação da metformina e do inibidor LY294002 no controle do processo de angiogênese em câncer de mama, por meio dos marcadores angiogênicos VEGF e HIF-1α.

Introdução: A síndrome dos ovários policísticos (SOP) é um distúrbio endócrino complexo com aspectos reprodutivos e metabólicos. Alterações do tecido adiposo participam da fisiopatologia da síndrome e anormalidades do metabolismo de ácidos graxos, bem como o estresse oxidativo, parecem ter papel importante nesse processo. Assim, tanto o exercício físico como a metformina são comumente recomendadas. O objetivo deste estudo foi avaliar a expressão de genes envolvidos com a síntese e oxidação dos ácidos graxos e com os níveis de estresse oxidativo no tecido adiposo de ratas com modelo experimental de SOP tratadas com metformina (Met), submetidas ou não a exercício físico em esteira (Ex). Métodos: Foram utilizadas 46 ratas, Wistar, que receberam no 2º dia de vida, uma única injeção subcutânea de propionato de testosterona (1,25 mg) para a indução de estado de estro permanente e óleo de sésamo (controles). Com 80 dias de idade iniciou-se o experimento e os animais foram divididos em 5 grupos: 1) Controle; 2) SOP ; 3) SOP+Met; 4) SOP+Ex; 5) SOP+Met+Ex.Os tratamentos tiveram duração de 6 semanas. Os animais foram sacrificados aos 120 dias de vida e foram retirados os depósitos das gorduras inguinal e mesentérica. Os depósitos de gordura inguinal e mesentérica foram utilizados para análise da expressão dos genes Acaca, Srebp1, Cpt1 e Cd36, por PCR quantitativo em tempo real, e dos níveis de estresse oxidativo pela dosagem das glutationas reduzida (GSH) e oxidada (GSSG). Adicionalmente, o tamanho dos adipócitos foi analisado por histomorfometria. Resultados: Na análise histomorfométrica dos adipócitos da gordura mesentérica, houve diminuição significativa no grupo SOP+Met+Ex em relação ao grupo SOP. O grupo SOP apresentou hipertrofia em relação ao grupo controle. A análise na gordura inguinal não apresentou nenhuma diferença entre os grupos.Não houve diferença estatística entre os grupos nas análises de expressão gênica das gorduras mesentérica e marrom. Não identificamos diferenças nas medidas de estresse oxidativo (razões GSH/GSSG ,GSH, GSH total, GSSG e razão GSH/GSSG) no tecido adiposo subcutâneo, mesentérico, perigonadal e marrom entre os depósitos de gordura dos animais em nenhum dos grupos avaliados / Introduction: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder, affecting both reproductive and endocrine systems. Changes in adipose tissue are involved in the pathophysiology of the syndrome, and abnormalities of fatty acid metabolism, as well as oxidative stress, appear to play an important role in this process. Thus, both physical exercise and metformin are commonly recommended. The objective of this study was to evaluate the expression of genes involved in the synthesis and oxidation of fatty acids and oxidative stress levels in adipose tissue of in an experimental model of PCOS using Wistar rats. Methods: 46 rats, Wistar, treated with a single subcutaneous injection of testosterone propionate (1.25 mg) for the induction of permanent estrus status (PCOS model) or sesame oil (controls) on the 2nd day of life. Experimental phase of the study was started when animals completed 80 days of age. They were divided into 5 groups: 1) Control; 2) PCOS; 3) PCOS+ Met; 4) PCOS + Ex; 5) PCOS + Met + Ex. Intervention lasted 6 weeks. Animals were sacrificed at 120 days of age and the deposits of inguinal and mesenteric fat were removed. Inguinal and mesenteric fat deposits were used to analyze the expression of Acaca, Srebp1, Cpt1 and Cd36 genes by quantitative realtime PCR and oxidative stress levels by reduced (GSH) and oxidized glutathione (GSSG). In addition, adipocyte size was analyzed by histomorphometry. Results: In the histomorphometric analysis of mesenteric fat adipocytes, there was a significant decrease in the PCOS + Met + Ex group in relation to the PCOS group. PCOS group presented hypertrophy in relation to the control group. Inguinal fat analysis did not present any differences between the groups. There was no statistical difference between groups in analyzes of gene expression of mesenteric and brown fats. We did not identify differences in oxidative stress measurements (GSH / GSSG, GSH, total GSH, GSSG and GSH / GSSG ratio) in the subcutaneous, mesenteric, perigonadal and brown adipose tissue among the fat deposits of the animals in any of the evaluated groups

Abstract Endometrial cancer (EC) is the fifth most common female cancer worldwide and its incidence is increasing. The prognosis of EC is fairly good. Histologically, ECs are categorized into endometrioid and non-endometrioid subtypes. Lately, the idea of repurposing existing medications for the prevention and co-treatment of EC has evoked interest in the scientific community. The results of preclinical studies involving various forms of antidiabetic medication (ADM) such as metformin, or cholesterol-lowering statins have been promising. In the previous epidemiological studies, the results of metformin and/or statin use and the risk and prognosis of EC have indicated either neutral or beneficial effects. At least some of these studies have several limitations, including a potential for several types of bias, and missing information on the dose and timing of medication, cancer-specific mortality or the histology of EC. The aim of this study was to find reliable further evidence on whether the use of metformin or statins could have beneficial effects on the risk and prognosis of EC in women with type 2 diabetes (T2D). Endometrioid and non-endometrioid EC were analyzed separately based on data from the Finnish Cancer Registry (FCR). In our study cohort of 92 366 women obtained from a nationwide diabetes database (FinDM) (1996 to 2011), the incidence rates of endometrioid (n = 590 cases) and non-endometrioid (n = 57 cases) EC were not found to differ between metformin users and users of other forms of oral ADM when adjusted for age, duration of T2D and use at any time of other forms of medication under study. We found insufficient evidence that metformin affects the prognosis of patients diagnosed with endometrioid (n = 1215) or non-endometrioid (n = 105) EC (1998 to 2011) after adjusting for year, age and stage at diagnosis of EC, and duration of T2D. However, in patients with endometrioid EC, mortality from other (predominantly cardiovascular) causes of death was decreased in metformin users compared with users of other types of oral ADM. Despite promising preclinical data, we were not able to confirm a beneficial effect of metformin use on the risk or prognosis of EC in women with T2D. In statin users, a lower risk of both EC subtypes and reduced cancer-specific mortality from non-endometrioid EC were observed. / Tiivistelmä Kohdun runko-osan syöpä on naisten viidenneksi yleisin syöpä, ja todettujen tapauksien määrä kasvaa. Syövän paranemisennuste on melko hyvä. Histologisesti syöpä jaetaan endometrioidi-muotoon ja ei-endometrioidi -muotoon. Alun perin muihin tarkoituksiin kehitettyjen lääkkeiden käyttö kohdun runko-osan syövän ehkäisyssä ja hoitoyhdistelmissä on ollut viime aikoina tieteellisen mielenkiinnon kohteena. Prekliinisten tutkimusten tulokset diabeteslääke metformiinin ja hyperkolesterolemian hoitoon käytettyjen statiinien osalta ovat olleet lupaavia. Aiemmissa epidemiologisissa tutkimuksissa metformiinin tai statiinien käytön vaikutukset kohdun runko-osan syövän riskiin ja ennusteeseen ovat olleet vaihtelevia. Osassa tutkimuksista on ollut ongelmia liittyen tilastollisten harhojen riskiin, puutteellisiin tietoihin lääkityksen kestosta ja kumulatiivisista annoksista sekä spesifisestä syöpäkuolleisuudesta ja syövän histologiasta. Kansalliseen diabetestietokantaan (FinDM) perustuvan tutkimuksemme tavoitteena oli selvittää, onko metformiinin tai statiinien käytöllä (Kelan lääkekorvaustilastot) kohdun runko-osan syövän riskiä vähentävää tai ennustetta parantavaa vaikutusta tyypin 2 diabetesta sairastavilla naisilla. Endometrioidit-syövät ja ei-endometrioidit -syövät analysoitiin erikseen Suomen Syöpärekisterin tietoihin perustuen. Kohortissamme (n = 92 366) ei todettu eroa endometrioidin (n = 590) tai ei-endometrioidin (n = 57) kohdun runko-osan syövän ilmaantuvuudessa metformiinia tai muita oraalisia diabeteslääkkeitä käyttävien naisten välillä (1996-2011), kun ikä, diabeteksen kesto ja muiden lääkitysten käyttö vakioitiin. Emme löytäneet näyttöä metformiinin käytön yhteydestä syöpäkuolleisuuteen endometrioidissa (n = 1 215) tai ei-endometrioidissa (n = 105) alatyypeissä verrattuna muihin diabeteslääkityksiin (1998-2011), kun ikä, syövän diagnoosivuosi ja levinneisyys sekä diabeteksen kesto vakioitiin. Endometrioidiin syöpään sairastuneilla metformiinia käyttävillä naisilla muu, valtaosalla sydän- ja verisuonitautiperäinen, kuolleisuus oli vähentynyt verrattuna muiden oraalisten diabeteslääkkeiden käyttäjiin. Aiemmista lupaavista tutkimustuloksista huolimatta emme todenneet metformiinilla olevan edullisia vaikutuksia kohdun runko-osan syövän kannalta. Statiinien käyttöön liittyi vähentynyt tämän syövän riski sekä vähentynyt syöpäkuolleisuus ei-endometrioidissa alatyypissä.

Submitted by CAMILA LEONEL DA SILVA null (camilaleonels_1@hotmail.com) on 2016-05-02T19:33:26Z No. of bitstreams: 1 TESE - Camila Leonel da Silva.pdf: 12839463 bytes, checksum: a432431aa8b8009a0183a4c0896c3e34 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-04T19:19:33Z (GMT) No. of bitstreams: 1 silva_cl_dr_sjrp.pdf: 12839463 bytes, checksum: a432431aa8b8009a0183a4c0896c3e34 (MD5) / Made available in DSpace on 2016-05-04T19:19:33Z (GMT). No. of bitstreams: 1 silva_cl_dr_sjrp.pdf: 12839463 bytes, checksum: a432431aa8b8009a0183a4c0896c3e34 (MD5) Previous issue date: 2016-04-23 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A transição epitélio-mesenquimal (EMT) é o processo pelo qual as células cancerosas a partir de tumores primários passam por uma conversão fenotípica para invadir e migrar, gerar metástases em tecidos ou órgãos distantes. Este processo pode ser induzido por fatores de crescimento, tais como Fator de Crescimento Transformante beta (TGF-β) e sua alta expressão tem sido implicada na angiogênese tumoral, na migração e invasão celular em muitos tipos de tumores. A expressão de ROCK-1 está associada com a malignidade dos tumores, enquanto a inibição desta molécula resulta em uma supressão significativa de metástases tumorais. A metformina, um fármaco utilizado no tratamento da diabetes, demonstrou inicialmente inibir a EMT e impedir o fenótipo mesenquimal pela repressão transcricional de pontos chave da regulação da EMT (ZEB1, TWIST1, SNAIL2, TGF-β) em células de câncer de mama. Os objetivos foram avaliar a expressão gênica e proteica de marcadores relacionados a metástase, em um estudo in vitro e in vivo, em linhagens de câncer de mama, após o tratamento com metformina, além do silenciamento gênico do TGF-β1 para inibição da transição epitélio-mesenquimal. Foi realizado a transfecção da linhagem celular metastática de tumor mamário canino CF41 de forma estável após a construção de um pequeno RNA de interferência para desenvolver derivados clonais que expressam níveis reduzidos de TGF-β1 (células TGF-β1sh). Este foi subsequentemente combinado com o tratamento com metformina, para analisar os efeitos sobre a migração de células, assim como a expressão dos marcadores de EMT E-caderina e N-caderina, quantificados através de imunofluorescência e do qRT-PCR. As linhagens mamárias humanas MCF-7 (não-metastática) e MDA-MB-231 (metastática) foram tratadas com metformina e inibidor Y27632, após a indução da EMT por TGF-β1 para examinar os efeitos sobre a migração destas células, bem como a expressão proteica dos marcadores ROCK-1, vimentina, E-caderina, CD44 e CD24 por imunocitoquímica. Em um estudo in vivo, as células não modificadas CF41 ou que expressam TGF-β1 shRNA foram injetadas na região inguinal de camundongos fêmea nude atímicos tratados com metformina. Os camundongos foram eutanasiados após o tratamento e os pulmões foram recolhidos para avaliação do número de metástases. As regiões metastáticas foram subsequentemente avaliadas pela expressão de N-caderina, E-caderina, vimentina e claudina-7 através da imuno-histoquímica. Foi possível avaliar que a taxa de migração e invasão foi menor em células TGF-β1sh, em comparação com as células parentais CF41 e esta inibição foi significativa quando combinado com o tratamento com metformina. As análises in vitro demonstraram que o tratamento com metformina reduziu a expressão de N-caderina e aumentou a expressão de E-caderina nas células CF41 e TGF-β1sh. Os resultados demonstram também que após a indução do TGF-β1 nas linhagens MCF-7 e MDA-MB-231 houve menor expressão das proteínas ROCK-1, vimentina, CD44 e CD24 em ambas as linhagens após tratamento com metformina e Y27632. Nas células MDA-MB-231 a expressão de E-caderina foi maior em todos os grupos de tratamento. O tratamento da linhagems MDA-MB-231 com metformina e Y27632 reduziu significativamente a invasão destas células. O estudo in vivo demonstrou que o tratamento com metformina reduziu o número de metástases pulmonares em animais portadores de tumores induzidos com as células TGF-β1sh. Houve diminuição da expressão de marcadores mesenquimais N-caderina e vimentina, e aumento da expressão de marcadores epiteliais E-caderina e claudina-7 nas metástases pulmonares. Assim, concluimos que este estudo confirma os benefícios do silenciamento do TGF-β1, além do tratamento com metformina e Y27632 como potenciais agentes terapêuticos em tumores de mama, bloqueando o processo de EMT e seu potencial metastático. / Epithelial mesenchymal transition (EMT) is the process by which cancer cells from primary tumors pass through a phenotypic conversion to invade and migrate, generating metastases in organs or tissues distant. This process can be induced by growth factors such as transforming growth factor beta (TGF-β) and its overexpression has been implicated in tumor angiogenesis, cell migration and invasion in many cancers. ROCK-1 expression is associated with the malignant character of tumors, while inhibiting this molecule results in a significant suppression of tumor metastasis. Metformin, a drug use for the treatment of diabetes, was previously shown to inhibit EMT by suppressing expression of key transcription factors in breast cancer cells. The aims were to evaluate the gene expression and protein expression of related markers metastasis, in a study in vitro and in vivo in breast cancer cell lines after treatment with metformin in addition to the gene silencing of TGF-β1 for inhibiting epithelial-mesenquimal transition. These aims were contemplated performing transfected of canine metastatic mammary tumor cell line CF41 with small interfering RNA constructs to develop clonal derivatives expressing reduced levels of TGF-β1 (TGF-β1sh cells). This was subsequently combined with metformin treatment, to look at effects on cell migration, as well as the expression of the EMT markers E-cadherin and N-cadherin, which were quantified by immunofluorescence and qRT-PCR. MCF-7 and MDA-MB-231 cell lines were treated with metformin and Y27632, after induction of EMT by TGF-β1, to examine the effects on cell migration as well as the protein expression of the ROCK-1 markers, vimentin, E-cadherin, CD44 and CD24 by immunocitochemistry. In an in vivo study, unmodified or TGF-β1 shRNA-expressing CF41 cells were injected in the inguinal region of nude athymic female mice that were treated with metformin. Mice were sacrificed after treatment and the lungs were collected to assess the number of metastases. Metastatic nodules were subsequently assessed for, N-cadherin, E-cadherin, vimentin and claudin-7 expression via immunohistochemistry. With the obtained results it was possible to assess the migration and invasion rate was lower in TGF-β1sh cells as compared to parental CF41 cells and this inhibition was significant when combined with metformin treatment. In vitro analyses demonstrated that metformin treatment reduced n-cadherin expression and increased E-cadherin expression in both CF41 and TGF-β1sh cells. After TGF-β1 induction in MDA-MB231 and MCF-7 cell lines, there was a lower protein expression of ROCK-1, vimentin, CD44 and CD24 in both cell lines after treatment with metformin and Y27632. In MDA-MB-231 cells, E-cadherin expression was increased in all treatment groups. Treatment of MDA-MB-231 cell line with metformin and Y27632 significantly reduced the invasion of these cells. In vivo studies demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-β1sh tumors. This paralleled a decreased expression of mesenchymal markers N-cadherin and vimentin, and increased expression of epithelial markers E-cadherin and claudin-7 in lung metastases.This study confirms the benefits of TGF-β1 silencing in addition to metformin and Y27632 as potential therapeutic agents in mammary tumors, by blocking EMT process and metastatic potential. / FAPESP: 2012/09778-1